The long-term goal of this project, which remains unchanged from the previous granting periods, is the study of pre-mRNA splicing in human cells.The main focus of this proposal will be the study of sequence elements in pre-mRNA that play a role in the selection of splice sites in a multiintron precursor. The mechanisms that determine splice site selection in constitutive and alternative splicing are not well understood and present one of the major unresolved questions in pre-mRNA splicing. This proposal will attempt to elucidate these mechanisms by the following experimental approaches: 1) Transcripts with a short internal exon and two introns, a model for a multiintron pre-mRNA, will be spliced in vitro. Preliminary results show that in such model transcripts the internal exon is either skipped or included in the spliced product depending on the sequence of the splice sites and the length of the internal exon. The details of the mechanism of splice site selection as exemplified by exon skipping/inclusion will be further elucidated. In particular, a hypothesis will be tested that the selection of splice sites depends on the interplay between the "strength" of the splice sites flanking the short internal exon and the length of this exon. Additional mechanisms of splice site selection such as "exon definition" and splice site competition win also be investigated. 2) The interactions of splicing factors with model pre-mRNAs mentioned above will be investigated. Formation of splicing complexes will be analyzed by native gel electrophoresis. The kinetics and nature of the interactions of splicing factors with splice sites will be determined by nuclease protection assays. The model transcripts will also be used to study the role of splicing factors in alternative splicing. A hypothesis will be tested that the relative concentrations of "housekeeping" splicing factors are responsible for alternative exon skipping or exon inclusion. We will also test whether factors that appear to be specific for certain alternatively spliced pre-mRNAs can induce alternative splicing in our model systems. 3) The patterns of splicing of model pre-mRNAs in vitro, in cell-free extracts, and in vivo in transiently and stably transfected cells will be compared. The goal of these experiments is to determine if coupling of transcription and splicing affects the patterns of splice site selection and if there are any additional mechanisms that control splice site selection in vivo.